Targeting the steroidogenic pathway for treating and/or preventing allergic diseases

ABSTRACT

The present invention relates to methods and compositions for treating and/or preventing allergic diseases or conditions by inhibiting one or more components of the steroidogenic pathway.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 14/160,747, filed Jan. 22, 2014, now U.S. Pat. No. 9,534,221, whichclaims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 61/755,311, filed Jan. 22, 2013,both of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under grant numbers P01HL 036577, and R01 AI-77609, awarded by the National Institutes ofHealth. The Government of the United States has certain rights in theinvention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named“Seq_Listing_2879-126_ST25”, has a size in bytes of 1 KB, and wasrecorded on Jan. 22, 2014. The information contained in the text file isincorporated herein by reference in its entirety pursuant to 37 CFR §1.52(e)(5).

FIELD OF INVENTION

The present invention generally relates to methods and compositions fortreating and/or preventing allergic diseases or conditions by inhibitingone or more components of the steroidogenic pathway including but notlimited to proteins, enzymes, receptors, and protein by-products of thesteroidogenic pathway.

BACKGROUND OF THE INVENTION

Steroid hormones, including glucocorticoids (GCs), play an importantrole in the regulation of the immune system (Chrousos, G. P., N. Engl.J. Med. 332, 1351-1362 (1995)). Endogenous glucocorticoid synthesis iscontrolled by the hypothalamic-pituitary-adrenal axis (Chrousos, G. P.,N. Engl. J. Med. 332, 1351-1362 (1995); Rhen, T., & Cidlowski, J. A., N.Engl. J. Med. 353, 1711-1723 (2005)) and is regulated by thetranscriptional control of steroidogenic enzymes of the cytochrome P450gene family (Mueller, M., et al. J. Exp. Med. 203, 2057-2062 (2006)).Corticosteroids have been used in treating allergic diseases due totheir anti-inflammatory activity (Barnes, P J. Br. J. Pharmacol.163:29-43 (2011)), but, somewhat paradoxically, increasing evidenceindicates that corticosteroids may also enhance disease pathogenesis byactivating and enhancing growth of CD4 T cells and inhibiting Th1cytokine production (Cima, I., Fuhrer, A., & Brunner, T. Immunol. Lett.106, 99-102 (2006)). Glucocorticoids amplified immune responses insteroid-insensitive CD8⁺ T cells (Ohnishi, H., et al. J. Allergy Clin.Immunol. 121, 864-871 (2008)). As well, the corticosteroids themselvesmay induce Th2 cytokine production while simultaneously suppressing theproduction of Th1 cytokines (Koya, T. et al. J. Immunol. 179, 2787-2796(2007)).

The inhibitory role of GCs on immune cells is well characterized (DeBosscher, K., et al. Endocr. Rev. 24, 488-522 (2003); De Bosscher, K, &Haegeman, G. Mol. Endocrinol. 23, 281-291 (2009)). GCs reduceinflammation through inhibition of NF-κB and by inducing the expressionof anti-inflammatory proteins including annexin 1 and MAPK phosphatase 1(Chrousos, G. P. N. Engl. J. Med. 332, 1351-1362 (1995)). GCs and othersynthetic derivatives have been used to treat a variety of diseases,including inflammatory diseases of the intestine and asthma (Barnes, PJ. Br. J. Pharmacol. 163:29-43 (2011); Faubion, W. A. Jr., et al.Gastroenterology 121, 255-260 (2001)). Although the anti-inflammatoryactivity of GCs is well described, accumulating evidence suggests thatGCs can also enhance immune cell activation, inducing gene transcriptionand promoting the pathogenesis of allergic diseases (Cima, I., et al. J.Exp. Med. 200, 1635-1646 (2004); Ohnishi, H., et al. J. Allergy Clin.Immunol. 121, 864-871 (2008)). Steroid hormones are mainly produced inthe adrenal glands, but other tissues also produce GCs through theinduction of steroidogenic enzymes (Chrousos, G. P. N. Engl. J. Med.332, 1351-1362 (1995); Payne, A. H. Biol. Reprod. 42, 399-404 (1990)).The intestinal mucosa contains steroidogenic enzymes such as cytochromeP450, family 11, subfamily A, polypeptide 1 (Cyp11a1) and synthesizespotent GCs which exhibit both an inhibitory and a co-stimulatory role onintestinal T cell activation (Cima, I., et al. J. Exp. Med. 200,1635-1646 (2004)).

Cyp11a1 (also known as P450scc) is a key regulator of steroid biogenesisas the first and rate-limiting enzyme in the steroidogenic pathway,converting cholesterol to pregnenolone (Pazirandeh, A., et al. FASEB J.13, 893-901 (1999)). Induction of the Cyp11a1 promoter by epidermalgrowth factor involves a ras/MEK1/AP-1-dependent pathway (Croft, M. etal. J. Exp. Med. 180, 1715-1728 (1994)). Cyp11a1 is expressed primarilyin the cortex of the adrenal gland, but testis, ovary, placenta, thymus,and intestine also express Cyp11a1 (Cima, I., et al. J. Exp. Med. 200,1635-1646 (2004); Pazirandeh, A., et al. FASEB J. 13, 893-901 (1999)).Activation of Cyp11a1 results in a spectrum of steroid hormones,including glucocorticoids that are known to play a role in T cellfunction (Mosmann, T. R., and Coffman, R. L. Annu. Rev. Immunol. 7,145-173 (1989); Seder, R. A. et al. J. Immunol. 148, 1652-1656 (1992)).Several of the gonadal steroids have been shown to have important immuneeffects on T cells that express their cognate receptors. T cells expressreceptors for androgen and estrogen and receptor activation can impactcytokine gene transcription. These studies have related gender bias todifferences in the response of CD4, CD8, and T regulatory cells (DeBosscher, K., et al. Endocr. Rev. 24, 488-522 (2003); De Bosscher, K, &Haegeman, Mol. Endocrinol. 23, 281-291 (2009)). T cells also expressmany of the steroid metabolic enzymes (De Bosscher, K., et al. Endocr.Rev. 24, 488-522 (2003)). Depletion of Cyp11a1 in mice or rabbitsresults in steroid deficiency, female external genitalia, and death(Shih, M. C., et al. Mol. Cell. Endocrinol. 336, 80-84 (2011); Pang, S.,et al. Endocrinology 131, 181-186 (1992); Yang, X., et al. Endocrinology132, 1977-1982 (1993)). In humans, mutations in the Cyp11a1 gene resultin a steroid hormone deficiency, causing a rare and potentially fatalform of lipoid congenital adrenal hyperplasia (Kim, C. J., et al. J.Clin. Endocrinol. Metab. 93, 696-702 (2008); Al Kandari, H., et al. J.Clin. Endocrinol. Metab. 91, 2821-2826 (2006)). Patients with aheterozygous or homozygous mutation of Cyp11a1 exhibit adrenalinsufficiency and sex reversal (Tajima, T., et al. J. Clin. Endocrinol.Metab. 86, 3820-3825 (2001); Parajes, S., et al. J. Clin. Endocrinol.Metab. 96, E1798-E1806 (2011)).

Transcription factors such as Steroidogenic Factor-1 (SF-1), ActivatorProtein 2 (AP-2), and several tissue-specific GATA family proteinsenhance the transcription of Cyp11a1 through interactions with AP-1,specificity Protein-1 (SP-1) and AP-2 (National Asthma Education andPrevention Program (National Heart Lung and Blood Institute) ThirdExpert Panel on the Management of Asthma. National Center forBiotechnology Information (U.S.). Expert panel report 3 guidelines forthe diagnosis and management of asthma. Bethesda, Md.: NationalInstitutes of Health National Heart Lung and Blood Institute; 2007). Inparticular, the GATA protein family plays an important role in theregulation of Cyp11a1 expression (Barnes, P J. Br. J. Pharmacol.163:29-43 (2011)). GATA binding elements have been identified in theCyp11a1 promoter and Cyp11a1 expression was decreased in GATA3-deficientmice (Wei, G, et al. Immunity 35, 299-311 (2011)). GATA4 significantlyupregulated Cyp11a1 expression in granulosa cells (Sher, N., et al. Mol.Endocrinol. 21, 948-962 (2007)). These results identify important eventsin the transcriptional regulation of Cyp11a1 that directly affectsteroid synthesis and release.

CD4 Th cells play a pivotal role in the induction and control ofallergic inflammation, including food allergy (Islam, S. A., & Luster,A. D. Nature Med. 18, 705-715 (2012)). In a mouse model of food allergy,allergen-specific CD4 T cells were activated in the mesenteric lymphnodes and recruited to the small intestine, resulting in increasedlevels of Th2 cytokines in the inflamed small intestine (Knight, A. K.,et al. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G1234-G1243(2007)).

In humans, allergen-specific Th2 CD4 T cells are essential in thedevelopment and maintenance of both type I IgE-mediated andnon-IgE-mediated food allergic responses. In patients with anaphylacticpeanut allergy, increased numbers of peanut-specific IL-5- andIL-4-producing Th2 cells are found in peripheral blood (Prussin, C., etal. J. Allergy Clin. Immunol. 124, 1326-1332 (2009)). In addition,peanut-specific T cell lines from individuals with peanut anaphylaxisprimarily produce Th2 cytokines (IL-4, IL-13) (DeLong, J. H., et al. J.Allergy Clin. Immunol. 127, 1211-1218 (2011)). Other food allergies werealso characterized by increased levels of Th2 cytokines; in patientswith milk-induced gastrointestinal diseases, milk-specific CD4 T cellsderived from the duodenal mucosa produce high levels of Th2 cytokines,especially IL-13 (Beyer, K., et al. J. Allergy Clin. Immunol. 109,707-713 (2002)).

Allergic asthma is a heterogeneous inflammatory disorder of the airwayscharacterized by chronic airway inflammation and airwayhyperresponsiveness (AHR) (Kim, H. Y., et al. Nat. Immunol. 11, 577-584(2011); Holgate, S. T. Nat Med. 18, 673-683 (2012)). Numbers ofCD8⁺IL-13⁺T cells are increased in asthmatics (Gelfand, E. W. andDakhama, A. J. Allergy Clin. Immunol. 117, 577-582 (2006)) and duringthe development of experimental asthma in mice (Hamelmann, E. et al. J.Exp. Med. 183, 1719-1729 (1996); Miyahara, N. et al. J. Immunol. 172,2549-2558 (2004); Miyahara, N. et al. J. Immunol. 174, 4979-4984(2005)). In an atopic environment rich in IL-4, these CD8⁺ T cellsmediate asthmatic responses (Koya, T. et al. J. Immunol. 179, 2787-2796(2007)). However, the mechanisms regulating the conversion of CD8⁺effector T cells from IFN-γ to pathogenic IL-13-producing effector cellshave not been defined.

Asthma has increased dramatically over the past 50 years and now affects5-10% of the population in many developed countries (Kim, H. Y., et al.Nat. Immunol. 11, 577-584 (2011)). National and international guidelinesrecommend the use of inhaled corticosteroids as the first step incontrolling airway inflammation and symptoms in persistent asthma(Holgate, S. T. Nat Med. 18, 673-683 (2012); Gelfand, E. W. and Dakhama,A. J. Allergy Clin. Immunol. 117, 577-582 (2006)). However, it has beendemonstrated that 45% of steroid-naive asthmatic patients do not respondto inhaled corticosteroids. Corticosteroid insensitivity has beenadopted as a principal criterion for characterizing asthma severity(Hamelmann, E. et al. J. Exp. Med. 183, 1719-1729 (1996)). Increasednumbers of CD8⁺ T cells, which are more resistant than CD4⁻ T cells tocorticosteroids (Miyahara, N. et al. J. Immunol. 172, 2549-2558 (2004);Miyahara, N. et al. J. Immunol. 174, 4979-4984 (2005)), have beendetected in steroid-insensitive asthmatics (Koya, T. et al. J. Immunol.179, 2787-2796 (2007)) and have correlated with lower lung function(LaVoie, H. A. and King, S. R. Exp. Biol. Med. 234, 880-907 (2009)). Theinventors and others also found that numbers of CD8⁺IL-13⁻ cells wereincreased in experimental asthma models in mice (Shih, M. C. et al. Mol.Endocrinol. 22, 915-923 (2008); National Asthma Education and PreventionProgram (National Heart Lung and Blood Institute) Third Expert Panel onthe Management of Asthma. National Center for Biotechnology Information(U.S.). Expert panel report 3 guidelines for the diagnosis andmanagement of asthma. Bethesda, Md.: National Institutes of HealthNational Heart Lung and Blood Institute; 2007, Guidelines for thediagnosis and management of asthma. Bethesda, Md.: National Institutesof Health National Heart Lung and Blood Institute; 2007) as a result oftheir activation by IL-4-producing CD4⁺ T cells (Martin, R. J. et al. J.Allergy Clin. Immunol. 119, 73-80 (2007)). CD8⁺ T cells can be polarizedto effector subsets with cytokine profiles similar to those found inCD4⁺ T cells (Li, L. B. et al. Blood 110, 1570-1577 (2007); Payne, A. H.Biol. Reprod. 42, 399-404 (1990); van Rensen, E. L. et al. Am. J.Respir. Crit. Care Med. 172, 837-841 (2005)). Both in vivo and in vitro,IL-4 is capable of triggering CD8⁺ T cell differentiation from apredominant IFN-γ-producing cell to one producing IL-13. However, themechanisms underlying this conversion of CD8⁺ T cells is unknown.

Transcriptional profiling identified Cyp11a1 transcripts as one of themost highly up-regulated during the differentiation of CD8⁻ Tlymphocytes to a Tc2 phenotype, that is, a CD8 T cell capable of IL-13production. This upregulation of Cyp11a1 in CD8⁺ T cells is similar tothe upregulation seen in CD4⁺ T cells in a peanut allergy model,suggesting that this enzyme is essential in CD4⁺ and CD8⁺ T cells forpro-allergic differentiation.

CD4⁺ T cell differentiation into Th2 cells with production of IL-4,IL-5, IL-9, and IL-13 has been shown to be critical for the developmentof altered airway responsiveness and eosinophilic airway inflammation inexperimental models of asthma (Samy, T. S. et al. Endocrinology 142,3519-3529 (2001); Pottratz, S. T. et al. J. Clin. Invest. 93, 944-950(1994)). In addition to CD4⁺ T cells, CD8⁺ T cells can be polarized toeffector subsets with cytokine profiles similar to those found in CD4⁺ Tcells (Payne, A. H. Biol. Reprod. 42, 399-404 (1990); van Rensen, E. L.et al. Am. J. Respir. Crit. Care Med. 172, 837-841 (2005)). It has beenpreviously demonstrated that there is an important role for type 2 (Tc2)CD8⁺ T cells in the development of experimental asthma (Slominski, A. etal. FEBSI 273, 2891-2901 (2006)) as a result of their activation byIL-4-producing CD4⁺ T cells (Martin, R. J. et al. J. Allergy Clin.Immunol. 119, 73-80 (2007)). Increased expression of BLT1 (leukotrieneB4 receptor) on the surface of CD8⁺ T cells leads to their increasedaccumulation in the lungs (Guidelines for the diagnosis and managementof asthma. Bethesda, Md.: National Institutes of Health National HeartLung and Blood Institute; 2007). Both human (Miyahara, N. et al. J.Immunol. 172, 2549-2558 (2004)) and mouse (Miyahara, N. et al. J.Immunol. 174, 4979-4984 (2005)) CD8⁻ T cells demonstrate aninsensitivity to corticosteroids not seen in CD4⁺ T cells, supportingthe notion that CD8⁺ T cells are at the root of the failure ofasthmatics to respond to corticosteroids and may be responsible forpersistent AHR and inflammation (Koya, T. et al. J. Immunol. 179,2787-2796 (2007)). In asthmatics, numbers of CD8⁺ T cells in the airwayshave correlated with lower airway function (LaVoie, H. A. and King, S.R. Exp. Biol. Med. 234, 880-907 (2009)).

Current therapies for allergic asthma have been fairly restricted withfew new drugs introduced into the clinic in the last decade. Inhaledcorticosteroids have remained the main anti-inflammatory agent forasthma. Indeed, upwards of 40-50% of asthmatics fail to respond toinhaled corticosteroids with changes in FEV1 (Hamelmann, E. et al. J.Exp. Med. 183, 1719-1729 (1996)). Moreover, corticosteroids may alsoenhance disease pathogenesis, especially amplifying responses in thesteroid-insensitive population of CD8⁻ T cells (Miyahara, N. et al.Nature Med. 10, 865-869 (2004)). Corticosteroids may induce Th2 cytokineproduction while suppressing the production of Th1 cytokines. Acombination of steroid insensitivity and plasticity of CD8⁺ T cells maybe major contributors to the failure of some patients to respond tocorticosteroids. CD8⁺ BLT1⁺IL-13⁺ CD8⁺ T cells have been proposed to bea primary cause of the airway inflammation and hyperresponsiveness seenin asthma (National Asthma Education and Prevention Program (NationalHeart Lung and Blood Institute) Third Expert Panel on the Management ofAsthma. National Center for Biotechnology Information (U.S.). Expertpanel report 3 guidelines for the diagnosis and management of asthma.Bethesda, Md.: National Institutes of Health National Heart Lung andBlood Institute; 2007, Guidelines for the diagnosis and management ofasthma. Bethesda, Md.: National Institutes of Health National Heart Lungand Blood Institute; 2007). However, the mechanism underlying theconversion of CD8⁺ T cells from IFN-γ-producing cells to IL-13 producingcells remains unclear.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a method of treating orpreventing an allergic disease in a subject who has, or is at risk ofdeveloping an allergic disease, comprising administering atherapeutically effective amount of a steroidogenic pathway inhibitor.

In one aspect, the allergic disease can be selected from an allergiclung disease, allergen-induced airway hyperresponsiveness,allergen-induced inflammation, rhinitis, asthma, allergic rhinitis, foodallergy, eosinophilic esophagitis, chronic urticaria, atopic dermatitis,occupational allergy, allergic conjunctivitis, hay fever, airborneallergic sensitivities, stinging insect allergy, hypersensitivitypneumonitis, eosinophilic lung diseases, inflammatory bowel disease,ulcerative colitis, and Crohn's disease.

In one aspect, the allergic disease is caused by one or moreproteinaceous allergens.

In another aspect, the subject has been sensitized to an allergen or isat risk of becoming exposed to an allergen.

In one aspect, the food allergy is peanut allergy.

In yet another aspect, the allergic disease is an allergic lung disease.

The steroidogenic pathway inhibitor can be selected from an antibody, anantisense molecule, an siRNA molecule, an shRNA molecule, a receptorantagonist, a chemical entity, a nucleotide, a peptide, and a protein.In one aspect, the steroidogenic pathway inhibitor inhibits one or moreenzymes, receptors or protein by-products of the steroidogenic pathway.In another aspect, the steroidogenic pathway inhibitor inhibitscytochrome P450 family 11 subfamily A polypeptide 1 (Cyp11A1). In stillanother aspect, the steroidogenic pathway inhibitor is aminoglutethimideor a Cyp11A1 siRNA or shRNA molecule. In yet another aspect, thesteroidogneic pathway inhibitor inhibits 3βHSD. In still yet anotheraspect, the steroidogenic pathway inhibitor is trilostane. In anotheraspect, the steroidogneic pathway inhibitor inhibits cytochrome P450family 11 subfamily β polypeptide 1 (Cyp11β1). In yet another aspect,the steroidogenic pathway inhibitor is metyrapone.

Another embodiment of the invention relates to a method of inhibitingT-cell pro-allergic differentiation in a subject comprisingadministering a therapeutically effective amount of a steroidogenicpathway inhibitor. In one aspect, the T-cell pro-allergicdifferentiation is CD4+ T-cells to Th2 and Th17 cell differentiation. Inyet another aspect, the T-cell pro-allergic differentiation is CD8+T-cells to Tc2 cell differentiation. In still another aspect, the T-cellpro-allergic differentiation is IL4-induced conversion of CD8+ T-cellsinto IL-13 secreting cells. In another aspect, the T-cell pro-allergicdifferentiation is IL-4 induced conversion of CD4+ T-cells into IL-13secreting cells.

Various embodiments of the invention are described below. However, theinvention is not limited to embodiments described in this summary, asinventions described in the description that follows are also expresslyencompassed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show Cyp11a1 expression in CD8⁺ T cells generated in thepresence of IL-2 or IL-2+IL-4. (A) Protocol for differentiation of CD8⁺T cells in IL-2 or IL-2+IL-4 in vitro. (B) Cyp11a1 mRNA expression asdetected by quantitative RT-PCR in CD8⁻ T cells differentiated in IL-2or IL-2+IL-4. (C) Cyp11a1 protein levels as detected by immunoblotanalysis and densitometry of autoradiographs in CD8⁺ T cellsdifferentiated in IL-2 or IL-2+IL-4. (D) Immunohistochemical stainingfor Cyp11a1 in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 in vitro(×200). Quantitative analysis was performed by counting Cyp11a1-positivecells under the microscope. Data (mean±SEM) are from at least 3independent experiments. **p<0.01 compared to the IL-2 group.

FIGS. 2A-C show Cyp11a1 enzymatic activity regulates the functionalconversion of CD8⁺ T cells from IFN-γ- to IL-13-producing cells. (A)Pregnenolone levels determined by ELISA in supernatants from CD8⁺ Tcells differentiated in IL-2 or IL-2+IL-4 in the presence or absence ofAMG (500 μM). **p<0.01 compared to the IL-2 group. ##p<0.01 compared tothe IL-2+IL-4 group. (B) Cyp11a1 protein levels detected by immunoblotanalysis and densitometry of autoradiographs in CD8⁺ T cellsdifferentiated in IL-2 or IL-2+IL-4 with 500 μM AMG. **p<0.01 comparedto the IL-2 group. ##p<0.01 compared to the IL-2+SIINFEKL group. (C)Flow cytometric analysis of cytokine expression in CD8⁺ T cellsdifferentiated in IL-2 or IL-2+IL-4 and treatment with differentconcentrations of AMG. Data are from at least 7 independent experiments.

FIGS. 3A-B show that a short hairpin RNA (shRNA) specific for Cyp11a1prevents the conversion of CD8⁺ T cells from IFN-γ- to IL-13-producingcells. (A) Representative flow cytometric analysis of Cyp11a1 expressionafter transfection with plasmids encoding a Cyp11a1 shRNA or a scrambledcontrol shRNA. For quantitative analysis of Cyp11a1-positive cells, dataare from at least 4 independent experiments. **p<0.01 compared toscramble shRNA group. (B) Representative flow cytometric analysis ofcytokine expression in CD8⁺ T cells after transfection. For quantitativeanalysis of Cyp11a1-positive cells, data are from at least 3 independentexperiments. **p<0.01 compared to scramble shRNA group.

FIG. 4 shows the lineage specific transcription factor expression inCD8⁺ T cells. T-bet and GATA3 expression detected by quantitative RT-PCRin CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 with or withoutSIINFEKL in the presence or absence of 500 μM AMG Data (mean±SEM) arefrom at least 8 independent experiments. **p<0.01 compared to the IL-2group. ##p<0.01 compared to the IL-2+SIINFEKL group.

FIGS. 5A-F show the treatment of CD8-deficient recipients with CD8⁺ Tcells differentiated in IL-2 and AMG (500 μM) fails to restore AHR andinflammation. (A) Experimental protocol. (B) Changes in airwayresistance (RL). (C) Cell composition in BAL fluid. (D) Cytokine levelsin BAL fluid. (E) Representative photomicrographs of lung histology(×200). Quantitative analysis of goblet cells was as described inMaterials and Methods. (F) Quantitation of Cyp11a1-positive cells in thelung. Data (mean±SEM) were from at least 6-10 mice. *p<0.05, **p<0.01compared to secondary challenged CD8-deficient recipients. #p<0.05,##p<0.01 compared to secondary challenged CD8-deficient recipients of5×10⁶ IL-2-differentiated CD8₊ T cells.

FIGS. 6A-D show Cyp11a1 is expressed in mouse jejunum. (A) Protocol forinduction of peanut allergy. (B) Cyp11a1 mRNA expression detected byquantitative RT-PCR in peanut sensitized and challenged vs. shamsensitized and peanut challenged mice. (C) Representativeimmunohistochemical staining for Cyp11a1 (×200). (D) Quantitation ofmucosal Cyp11a1-expressing cells. Results were from 3 independentexperiments; each experiment included 4 mice per group (n=12). *P<0.05,**P<0.01. PBS/PE, sham sensitized and peanut challenged; PE/PE, peanutsensitized and challenged.

FIGS. 7A-C show the inhibition of Cyp11a1 enzymatic activity does notimpact levels of Cyp11a1 protein and mRNA expression in the mousejejunum. (A) Pregnenolone levels were assessed in serum of mice. (B)Cyp11a1 mRNA expression in jejunum of mice treated with AMG or vehicle.(C) Quantitation of mucosal Cyp11a1-expressing cells. Results were from3 independent experiments; each experiment included 4 mice per group(n=12). *P<0.05, **P<0.01, n.s. not significant. PBS/PE, sham sensitizedand peanut challenged; PE/PE, peanut sensitized and challenged;PE/PE/AMG 20 mg/kg, peanut sensitized and challenged and treated withAMG at dose of 20 mg/kg.

FIGS. 8A-E show the inhibition of Cyp11a1 enzymatic activity in vivoreduces intestinal responses. (A) Kinetics of development of diarrheaafter treatment with AMG (Cyp11a1 inhibitor) vs. vehicle. (B) Scoresbased on the severity of clinical signs were assessed 30 minutes afteroral challenge. (C-D) Quantitation of mucosal mast cell and goblet cellnumbers in jejunum. (E) Plasma histamine levels were assessed within 30minutes of the last oral challenge. Results were from 3 independentexperiments; each experiment included 4 mice per group. *P<0.05,**P<0.01, #P<0.001.

FIGS. 9A-B show the effects of Cyp11a1 inhibition on cytokine andlineage-specific transcription factor expression in the mouse jejunum.(A) IFNG, IL4, IL13, and IL17A mRNA expression in jejunum of micetreated with AMG or vehicle. (B) Th1, Th2, and Th17 transcriptionfactors T-bet, GATA3, and RORγt expression in jejunum of mice treatedwith AMG or vehicle. Results were from 3 independent experiments (n=12).*P<0.05, **P<0.01, n.s. not significant.

FIGS. 10A-F show the inhibition of Cyp11a1 enzymatic activity suppressesthe differentiation of naive CD4 T cells into Th2 and Th17 cells withoutaffecting lineage-specific transcription factor and Cyp11a1 expression.(A). Relative Cyp11a1 expression in naive CD4 T cells differentiated invitro into Th1, Th2, and Th17 cells from spleen of naive TCR-transgenicmice (OT II mice) determined by real time PCR. (B). Cyp11a1 mRNAexpression in polarized CD4 T cells in the presence of AMG or vehicle.(C). Western blot analysis of Cyp11a1 protein in polarized Th1, Th2, orTh17 cells treated with AMG or vehicle. (D). Pregnenolone levels wereassessed in supernatants of cultured CD4 T cells under Th1, Th2, andTh17 polarizing conditions. (E) Cytokine levels in supernatants ofcultured CD4 T cells treated with inhibitor or vehicle under Th1, Th2,and Th17 polarizing conditions. (F) Th1, Th2, and Th17 cytokine andlineage-specific transcription factor mRNA expression in polarized Th1,Th2, or Th17 cells treated with the inhibitor or vehicle. The data shownare from 3 independent experiments. *P<0.05, **P<0.01, #P<0.001, n.s.not significant.

FIGS. 11A-D shows shRNA-mediated silencing of Cyp11a1 regulates levelsof IL-13 without affecting levels of GATA3 transcripts in Th2 T cells.(A) Cyp11a1 mRNA expression in shRNA-transduced Th2 cells. (B)Pregnenolone levels were assessed in supernatants of cultured Th2 cellstransduced with Cyp11a1 or luc shRNA. (C) Levels of IL4, IL13, and GATA3mRNA expression in cultured Th2 cells transduced with Cyp11a1 or lucshRNA. (D) Levels of IL-4 and IL-13 in supernatants of cultured Th2cells transduced with Cyp11a1 or luc shRNA. Results were from 3independent experiments. *P<0.05, **P<0.01.

FIGS. 12A-E shows the decreased mast cell infiltration in the intestinalwall of PE/PE mice treated with AMG Intestinal mucosa mast cells werequantified in jejunum using chloroacetate esterase staining.Representative sections of (A) PBS/PE/vehicle mice; (B) PE/PE/vehiclemice; (C) PE/PE/AMG (5 mg/kg) mice; (D) PE/PE/AMG (10 mg/kg) mice; and(E) PE/PE/AMG (20 mg/kg) mice. Magnification ×400.

FIG. 13A-E shows the decreased numbers of goblet cells in intestinalepithelium of sensitized and challenged mice treated with AMG Gobletcells were identified by PAS staining 24 hrs after the last challenge.Representative sections of (A) PBS/PE/vehicle mice, (B) PE/PE/vehiclemice, (C) PE/PE/AMG (5 mg/kg) mice, (D) PE/PE/AMG (10 mg/kg) mice, and(E) PE/PE/AMG (20 mg/kg) mice. Magnification ×200.

FIG. 14A-B shows the treatment with AMG had no effect on serumimmunoglobulin production in peanut sensitized and challenged mice.Serum levels of peanut-specific IgE (FIG. 14A), IgG1 (FIG. 14B), andIgG2a (FIG. 14B) were assessed by ELISA 24 hrs after the last challengeand expressed as optical density of diluted serum as described inMethods. Results were obtained from 3 individual experiments with 4 miceper group. #P<0.001, “n.s.” indicates “not significant”.

FIG. 15 shows a schematic representation of the major mammaliansteroidogenic pathway(s) (from Simard et al., Endocrine Rev, June 2005,26(4):525-82).

FIG. 16 shows an overview of steroidogenesis.

FIG. 17 shows representative photomicrographs of immunohistochemicalstaining for Cyp11a1-positive cells in the lung (×200). The lungs werefrom secondary challenged CD8-deficient recipients, secondary challengedCD8-deficient recipients of 5×10⁶ IL-2-differentiated CD8⁺ T cells, andsecondary challenged CD8-deficient recipients of 5×10⁶IL-2-AMG-differentiated CD8^(|) T cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to methods for the prevention and/ortreatment of an allergic disease or condition, as well as methods ofinhibiting T-cell pro-allergic differentiation in subjects who have orare risk of developing an allergic disease or condition. The inventionincludes administration of a therapeutically effective amount of asteroidogenic pathway inhibitor. The invention includes the use of acomposition comprising a steroidogenic pathway inhibitor as well as thecomposition itself. The invention also includes kits that contain one ormore steroidogenic pathway inhibitors.

Steroid hormones play a critical role in the differentiation,development, growth, and physiological function of most vertebratetissues. The major pathways of steroid hormone synthesis are wellestablished, and the sequence of the responsible steroidogenic enzymeshas been elucidated (Simard et al., Endocrine Rev. June 2005,26(4):525-82; see also FIGS. 15 and 16). Many of the enzymes of thesteroidogenic pathway are localized to the smooth endoplasmic reticulum(ER) with the exceptions of P450 scc (i.e. P450 cholesterol side-chaincleavage; CYP11A1), P450c11 (CYP11B1), and aldosterone synthase(CYP11B2) (Simard et al., Endocrine Rev. June 2005, 26(4):525-82).

Many inhibitors of the various components of the steroidogenic pathwayare known including but not limited to aminoglutethimide (inhibitsCyp11A1), trilostane (inhibits β3HSD—also known as 3-β-HSD; or3-β-hydroxysteroid dehydrogenase/Δ-5-4 isomerase) and metyrapone(inhibits Cyp11β1).

In the present invention, the inventors demonstrate the role of Cyp11a1in controlling IL-4-mediated CD8⁺ T cell conversion in vitro and invivo. The inventors demonstrate that mRNA transcript levels, proteinlevels, and the enzyme activity of Cyp11a1 in CD8⁺ T cells are allincreased surprisingly following differentiation in the presence ofIL-2+IL-4 compared to IL-2 alone. Further, the Cyp11a1 enzyme inhibitoraminoglutethimide (AMG) or knock-down of Cyp11a1 protein levels using aspecific shRNA (small or short hairpin RNA), blocked the functionalconversion of CD8⁺ T cells from IFN-γ- to IL-13-producing cells.Expression of the lineage-specific transcription factors T-bet or GATA3was not affected by inhibition of Cyp11a1 activity, indicating that itwas downstream of expression of these master regulatory transcriptionfactors. Adoptive transfer of AMG-treated CD8⁺ T cells, in contrast tountreated CD8⁺ T cells, failed to restore AHR and inflammation insensitized and challenged CD8-deficient mice. The inventors demonstratefor the first time Cyp11a1 as a key regulator of CD8⁺ pro-allergic Tc2cell differentiation and plasticity.

CD8⁺ T cells have been primarily associated with production of IFN-γ;however, in the presence of IL-4, CD8⁺ T cells were skewed todifferentiate into IL-13-producing cells. This differentiation wasassociated with increases in GATA3 and decreases in T-bet expression andwas dependent on antigen signaling through the T cell receptor. AlthoughIL-4 triggered the up-regulation of Cyp11a1 mRNA, protein, and enzymaticactivity, the function of Cyp11a1 enzymatic activation was downstream ofGATA3 and T-bet transcriptional events as their expression levels wereunaffected by blocking Cyp11a1 activity with AMG Since addition ofSIINFEKL (SEQ ID NO:1) was also required for IL-13 cytokine production(FIG. 2C), it thus appeared that T-cell receptor signaling andactivation of Cyp11a1 enzymatic activity were both required for thelater stages in CD8 skewing to a Tc2 (IL-13) phenotype.

Taken together, the data presented herein establish for the first timethat the steroidogenic enzyme Cyp11a1 plays a direct role in thepolarization of CD8⁺ T cells from an IFN-γ- to an IL-13-producingeffector cell and, as a result, is a critical regulator of thedevelopment of lung allergic responses. Cyp11a1 thus represents apivotal enzyme linking steroidogenesis in T cells to pro-allergicdifferentiation pathways.

The inventors also demonstrate that peanut sensitization and challengenot only results in inflammatory and cytokine changes in the smallintestine but that mRNA, protein, and enzymatic activity levels of thesteroidogenic enzyme Cyp11a1 are also markedly elevated. Administrationof an inhibitor of Cyp11a1 enzymatic activity, AMG prevented developmentof allergic diarrhea and accumulation of inflammatory cells in the smallintestine in a dose-dependent manner. Levels of serum pregnenolone werereduced in parallel. AMG treatment decreased IL13 and IL17 mRNAexpression in the small intestine without impacting Cyp11a1 mRNA orprotein levels. In vitro, the inhibitor decreased levels of IL13 andIL17 mRNA in polarized Th2 and Th17 CD4 T cells, respectively, withoutaffecting levels of GATA3, RORγt, or the polarization of Th1 cells,IFNG, and T-bet expression. The importance of Cyp11a1 was furtherdemonstrated using shRNA-mediated silencing of Cyp11a1 in polarized Th2CD4 T cells which resulted in significantly decreased levels of IL-4 andIL-13 mRNA and protein. These data demonstrate that Cyp11a1 played animportant role in the development of peanut allergy through its effectson steroidogenesis, a critical pathway in CD4⁻ T cell Th2differentiation.

The inventors demonstrate for the first time that levels of Cyp11a1protein and mRNA are increased in the jejunum of sensitized andchallenged mice. In parallel, enzymatic activity is increased asdemonstrated by increased levels of pregnenolone in the serum ofsensitized and challenged mice. The inventors also demonstrate thatCyp11a1 enzymatic activity is essential for induction of peanut allergyusing an inhibitor, AMG Administration of this inhibitor during the oralchallenge phase, after sensitization, results in significantly lowerserum pregnenolone levels and reduces the incidence and severity ofdiarrhea and intestinal inflammation (mast cell accumulation and gobletcell metaplasia), accompanied by decreases in IL13 and IL17A mRNA in theintestine. The inhibitor did not alter the development of specificantibodies, including peanut-specific IgE, likely because sensitizationwas completed prior to treatment in the challenge phase. Althoughadministration of the inhibitor in vivo could not identify specifictarget cells, these data demonstrated for the first time that Cyp11a1functions as a key regulator of the development of peanut-inducedallergic responses.

The data described in the Examples presented herein demonstrate thatinhibition of Cyp11a1 significantly reduces CD4⁺ Th2 and Th17 cytokineproduction in vivo. Interestingly, the inhibitor does not affectexpression of the Th1, Th2, and Th17 lineage-specific transcriptionfactors T-bet (Th1-specific T box transcription factor), GATA3(GATA-binding factor 3), or RORγt (RAR-related orphan receptor gamma t).The results support that suppression of Th2 and Th17 cytokine productionis not mediated through effects on lineage-specific transcription factorexpression but on cytokine transcription. The primary action of Cyp11a1enzymatic activity manifests downstream of these lineage-specifictranscription factors.

Further, the function of Cyp11a1 in CD4 T cells, Th1, Th2, and Th17polarization was monitored in vitro in the presence of AMG The highestlevels of Cyp11a1 protein and enzymatic activity were detected inpolarized Th2 cells, with significantly lower levels in Th17 cells, andvirtually no activity in Th1 cells. The inhibitor decreased IL-13cytokine production in polarized Th2 cells; however, IFN-γ productionwas not affected by the inhibitor in polarized Th1 cells. Similar to thein vivo data, the inhibitor did not affect GATA3 mRNA expression inpolarized Th2 cells nor levels of T-bet or RORγt in polarized Th1 andTh17 cells, respectively. Thus, inhibition of Cyp11a1 enzymatic activityimpaired CD4 Th2 and Th17 cell differentiation, which in turn decreasedproduction of the Th2 cytokine (IL-13) and Th17 cytokine (IL-17A) andthese effects were mediated downstream of their respective and essentiallineage-specific transcription factors.

Additionally, Cyp11a1 mRNA was silenced in cultured Th2 CD4 T cellsusing a short hairpin RNA (shRNA) to demonstrate that the results withAMG were specific to inhibition of Cyp11a1. During Th2 polarization,cells were transduced with retrovirus expressing Cyp11a1-targeted shRNAor control (luc) shRNA and activated under Th2 conditions. Cyp11a1 shRNAdecreased the expression of Cyp11a1 mRNA levels by 58%±5.2% andenzymatic activity of Cyp11a1, monitoring pregnenolone levels, wasreduced by 47%±4.5%. Levels of Th2 cytokine (IL4, IL13) mRNA and proteinwere decreased upon transduction of Cyp11a1 shRNA. As we observed withCyp11a1 inhibition in vivo and in vitro with AMG levels of GATA3 mRNAremained unaffected after silencing of Cyp11a1. These data confirmed invivo and in vitro AMG inhibition data, demonstrating that Cyp11a1critically regulates Th2 cell differentiation and cytokine production.

These studies demonstrate for the first time that activation of thesteroidogenic enzyme Cyp11a1 plays a critical role in the development ofintestinal allergic responses through its effects on CD4⁺ Th2polarization and IL-13 production. Cyp11a1 thus is a novel target forthe regulation and treatment of peanut-induced allergy.

According to the present invention, allergic diseases and/or conditions,include but are not limited to pulmonary conditions such as allergiclung diease, allergic rhinitis, asthma, airway hyperresponsiveness,allergen-induced airway hyperresponsiveness and hay fever as well asother allergic conditions including but not limited to a food allergy,allergen-induced inflammation, eosinophilic esophagitis, chronicurticaria, atopic dermatitis, occupational allergy, allergicconjunctivitis, airborne allergic sensitivities, stinging insectallergy, hypersensitivity pneumonitis, eosinophilic lung diseases,inflammatory bowel disease, ulcerative colitis, Crohn's disease and drugallergies. Symptoms of the allergies, including but not limited todiarrhea and intestinal inflammation as well as asthma and airwayhyperresponsiveness, is apparently or obviously, directly or indirectlytriggered by an allergen to which a subject has previously beensensitized. In one aspect, the allergic disease or condition can becaused by one or more proteinaceous allergens. Sensitization to anallergen refers to being previously exposed one or more times to anallergen such that an immune response is developed against the allergen.Responses associated with an allergic reaction, including but notlimited to histamine release, edema, vasodilatation, bronchialconstriction, airway inflammation, airway hyperresponsiveness, asthma,allergic rhinitis (hay fever), nasal congestion, sneezing, running nose,skin rash, diarrhea including acute allergic diarrhea and intestinalinflammation), typically do not occur when a naive subject is exposed tothe allergen for the first time, but once a cellular and humoral immuneresponse is produced against the allergen, the subject is “sensitized”to the allergen. Allergic reactions then occur when the sensitizedindividual is re-exposed to the same allergen (e.g., an allergenchallenge). Once a subject is sensitized to an allergen, the allergicreactions can become worse with each subsequent exposure to theallergen, because each re-exposure not only produces allergic symptoms,but further increases the level of antibody produced against theallergen and the level of T cell response against the allergen.

According to the present invention, inflammation is characterized by therelease of inflammatory mediators (e.g., cytokines or chemokines) whichrecruit cells involved in inflammation to a tissue. A condition ordisease associated with allergic inflammation is a condition or diseasein which the elicitation of one type of immune response (e.g., aTh2-type immune response) against a sensitizing agent, such as anallergen, can result in the release of inflammatory mediators thatrecruit cells involved in inflammation in a subject, the presence ofwhich can lead to tissue damage and sometimes death. A Th2-type immuneresponse is characterized in part by the release of cytokines whichinclude IL-4, IL-5, and IL-13. A TH17-type response is characterized bythe release of IL-17. The present invention is particularly useful fortreating allergen-induced food allergies (such as peanut allegories) andairway hyperresponsiveness and airway inflammation, including,allergen-induced asthma and rhinitis.

Accordingly, various embodiments of the present invention includetreating a subject that has been sensitized to an allergen and has beenor is at risk of becoming exposed to the allergen. In other embodiments,the present invention includes preventing an allergic disease orcondition in a subject at risk of becoming exposed to the allergen. Suchallergens can be related to a food, a plant, a gas, a pathogen, a metal,a glue or a drug. Examples of food allergens include but are not limitedto groundnuts such as peanuts; nuts from trees including Brazilian nuts,hazelnuts, almonds, walnuts; fruit, milk, eggs, fish, shellfish, wheat,or gluten. Examples of plant allergens include but are not limited topollen, trees, grass, weeds, ragweed, poison Oak or poison ivy. Examplesof gas allergens include but are not limited to environmental tobaccosmoke, and carbon monoxide. Examples of pathogen allergens include butare not limited to mold, viruses or bacteria. Examples of metalallergens include but are not limited to lead, nickel, chromate, orcobalt. Examples of drug allergens include but are not limited topenicillin, sulfur, or aspirin. Additional allergens include but are notlimited to latex, dust mites, pet dander (skin flakes), droppings fromcockroaches, rodents and other pests or insects.

According to the present invention, “airway hyperresponsiveness” or“AHR” refers to an abnormality of the airways that allows them to narrowtoo easily and/or too much in response to a stimulus capable of inducingairflow limitation. AHR can be a functional alteration of therespiratory system resulting from inflammation in the airways or airwayremodeling (e.g., such as by collagen deposition). Airflow limitationrefers to narrowing of airways that can be irreversible or reversible.Airflow limitation or airway hyperresponsiveness can be caused bycollagen deposition, bronchospasm, airway smooth muscle hypertrophy,airway smooth muscle contraction, mucous secretion, cellular deposits,epithelial destruction, alteration to epithelial permeability,alterations to smooth muscle function or sensitivity, abnormalities ofthe lung parenchyma and infiltrative diseases in and around the airways.Many of these causative factors can be associated with inflammation. AHRcan be triggered in a patient with a condition associated with the abovecausative factors by exposure to a provoking agent or stimulus. Suchstimuli include, but are not limited to, an allergen.

According to the present invention, treatment of a subject having anallergic disease and/or condition can commence as soon as it isrecognized (i.e., immediately) by the subject or by a clinician that thesubject has been exposed or is about to be exposed to an allergen.Additionally, preventing an allergic disease or condition can commenceprior to the subject being exposed to an allergen. Treating the subjectand/or preventing an allergic disease or condition in the subject, cancomprise administering a composition including but not limited to asmall molecule inhibitor, an antibody, a chemical entity, a nucleotide,a peptide, a protein, an antisense molecule, and siRNA molecule, andshRNA molecule that inhibits one or more proteins, and/orprotein-by-products, enzymes, and/or receptors of the steroidogenicpathway. Inhibiting a component of the sterodogenic pathway includesboth direct inhibition of the components as well as inhibition of theexpression of the one or more components of the pathway. Inhibition ofone or more components of the steroidogenic pathway can be by anymechanism, including, without limitations, decreasing activity of one ormore components, increasing inhibition of one or more of the components,degradation of one or more of components, a reduction or elimination ofexpression of one or more components and combinations thereof. Bindingto one or more component to prevent its wild-type enzymatic activity forexample, including competitive and noncompetitive inhibition, inhibitingtranscription, and regulating expression can also inhibit the component.These inhibitors can also reduce expression of CD4⁺ and CD8⁺ T cellproliferation and have the ability to suppress Th2 differentiationand/or Th17 differentiation.

The present invention also relates to a method of inhibiting T-cellpro-allergic differentiation in a subject by administering to thesubject a therapeutically effective amount of a steroidogenic pathwayinhibitor. In one aspect, the T-cell pro-allergic differentiation isCD4+ T-cells to Th2 and Th17 cell differentiation. In another aspect,the T-cell pro-allergic differentiation is CD8+ T-cells to Tc2 celldifferentiation. The T-cell pro-allergic differentiation can be IL-4induced conversion of CD8+ T-cells into IL-13 secreting cells. In stillanother aspect, the T-cell pro-allergic differentiation can be IL-4induced conversion of CD4+ T-cells into IL-13 secreting cells.

In accordance with the present invention, acceptable protocols toadminister a composition including the route of administration and theeffective amount of a composition to be administered to a subject can bedetermined by those skilled in the art. The composition of the presentinvention can be administered in vivo or ex vivo. Suitable in vivoroutes of administration can include, but are not limited to, aerosol,oral, nasal, inhaled, topical, intratracheal, transdermal, rectal, orparenteral routes. Preferred parenteral routes can include, but are notlimited to, subcutaneous, intradermal, intravenous, intramuscular, orintraperitoneal routes.

In one embodiment, the method of treating and/or preventing an allergicdisease and/or condition or inhibiting T-cell pro-allergicdifferentiation can comprise administering a therapeutically effectiveamount of a composition comprising a compound that interacts with aregulator of a component of the steroidogenic pathway including but notlimited to Cyp11A1 mRNA expression or Cyp11A1 protein expression. In oneaspect, the regulator is an inhibitor of the steroidogenic pathway,including but not limited to an antibody, an antisense molecule, ansiRNA molecule, an shRNA molecule, a receptor antagonist, a chemicalentity, a nucleotide, a peptide and a protein. In one aspect, thesteroidogenic pathway inhibitor inhibits one or more enzymes, receptors,or protein by-products of the steroidogenic pathway. In a preferredembodiment, the steroidogenic pathway inhibitor inhibits Cyp11A. Thisinhibitor can be aminoglutethimide, a Cyp11A siRNA molecule or a Cyp11AshRNA molecule. In other aspect, the steroidogenic pathway inhibitorinhibits 3βHSD and can be triostane. In still another aspect, thesteroidogenic pathway inhibitor inhibits Cyp11β1 (cytochrome P450 family11 subfamily β polypeptide 1) and can be metyrapone.

According to the methods of the present invention, a therapeuticallyeffective amount of a steroidogenic pathway inhibitor or a compositioncomprising a steroidogenic pathway inhibitor that is administered to asubject, comprises an amount that is capable of inhibiting expressionand/or activity of one or more components of the steroidogenic pathway(mRNA and/or protein) without being toxic to the subject. An amount thatis toxic to a subject comprises any amount that causes damage to thestructure or function of a subject (i.e., poisonous).

The invention also includes kits that contain one or more steroidogenicpathway inhibitors.

In addition, according to the present invention, the composition as wellas the kits of the present invention, can comprise a pharmaceuticallyacceptable excipient. According to the present invention, thecomposition, may be administered with a pharmaceutically acceptablecarrier, which includes pharmaceutically acceptable excipients and/ordelivery vehicles, for delivering the agent to a subject (e.g., aliposome delivery vehicle). As used herein, a pharmaceuticallyacceptable carrier refers to any substance suitable for delivering atherapeutic composition useful in the method of the present invention toa suitable in vivo or ex vivo site. Preferred pharmaceuticallyacceptable carriers are capable of maintaining the composition of thepresent invention in a form that, upon arrival of the composition to atarget cell, the composition is capable of entering the cell andinhibiting one or more components of the steroidogenic pathway (mRNAand/or protein) in the cell. Suitable excipients of the presentinvention include excipients or formularies that transport or helptransport, but do not specifically target a nucleic acid molecule to acell (also referred to herein as non-targeting carriers). Examples ofpharmaceutically acceptable excipients include, but are not limited towater, phosphate buffered saline, Ringer's solution, dextrose solution,serum-containing solutions, Hank's solution, other aqueousphysiologically balanced solutions, oils, esters, glycols andcombinations thereof. Aqueous carriers can contain suitable auxiliarysubstances required to approximate the physiological conditions of therecipient, for example, by enhancing chemical stability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate,sodium chloride, sodium lactate, potassium chloride, calcium chloride,and other substances used to produce phosphate buffer, Tris buffer, andbicarbonate buffer. Auxiliary substances can also include preservatives,such as thimerosal, m- or o-cresol, formalin and benzol alcohol.Compositions of the present invention can be sterilized by conventionalmethods and/or lyophilized.

According to the methods of the present invention, the subject can beany animal subject, and particularly, in any vertebrate mammal,including, but not limited to, primates, rodents, livestock or domesticpets. Preferred mammals for the methods of the present invention includehumans.

The following examples are provided for illustrative purposes, and arenot intended to limit the scope of the invention as claimed herein. Anyvariations which occur to the skilled artisan are intended to fallwithin the scope of the present invention. All references cited in thepresent application are incorporated by reference herein to the extentthat there is no inconsistency with the present disclosure.

EXAMPLES

Examples 1-6 demonstrate the role of Cyp11a1 in controllingIL-4-mediated CD8⁺ T cell conversion in vitro and in vivo.

Materials and Methods for Examples 1-6

Animals

OT-1 TCR transgenic (OT-1) mice and homozygous CD8-deficient mice werebred in the animal facility at National Jewish Health (Denver, Colo.).OT-1 mice (C57BL/6 strain) express a transgenic TCR specific forSIINFEKL peptide (ovalbumin (OVA)₂₅₇₋₂₆₄). CD8-deficient mice weregenerated by targeting the CD8⁺-chain gene in C57BL/6 mice (Oka, H. etal. Cell. Immunol. 206, 7-15 (2000); Sundrud, M. S. and Nolan, M. A.Curr. Opin. Immunol. 22, 286-292 (2010)). Animal experiments in thisstudy were conducted under a protocol approved by the InstitutionalAnimal Care and Use Committee of National Jewish Health.

CD8⁺ T Cell Culture

CD8⁺ effector memory T cells were generated in vitro as previouslydescribed (Miyahara, N. et al. J. Immunol. 172, 2549-2558 (2004);Miyahara, N. et al. J. Immunol. 174, 4979-4984 (2005)). In brief,mononuclear cells (MNCs) were processed from the spleens of OT-1 micefollowed by stimulation of 1 μg/ml SIINFEKL peptide (SEQ ID NO:1) wasused to stimulate cells for 1.5 hours. Two days after culture, livingcells were re-isolated using histopaque and cultured in complete RPMI1640 medium that contained recombinant mouse IL-2 (20 ng/ml) (R&D,Minneapolis, Minn.) or IL-2+IL-4 (20 ng/ml) (Peprotech, Rocky Hill,N.J.). For some experiments, AMG was added into the medium together withIL-2 or IL-2+IL-4. Medium with cytokines was changed every day for afurther 4 days. The cells were then re-stimulated with 1 μg/ml SIINFEKL(SEQ ID NO:1) in medium containing 2 μM monensin (Calbiochem, La Jolla,Calif.) for 4 hours.

RNA Preparation and Analysis

Total RNA was extracted from 5×10⁶ differentiated CD8⁺ T cells using theRNeasy Mini kit (Qiagen, Valencia, Calif.). 1 μg of total RNA wasconverted into cDNA using iScript cDNA Synthesis kit (Bio-Rad, Hercules,Calif.). Quantitative RT-PCR was performed using Cyp11a1 primers andprobe obtained from Applied Biosystems (Cat:Mm00490735_m1). Fold-changeswere determined using the 2^(ΔΔCt) method, with normalization toexpression of mouse GAPDH.

ELISA for Pregnenolone Measurements

CD8⁺ T cells generated in the presence of IL-2, IL-2+IL-4, IL-2+AMG orIL-2+IL-4+AMG were cultured in 6-well plates at 5×10⁶/ml for 24 hours.Supernatants were collected. Pregnenolone levels were measured using thePregnenolone ELISA kit (ALPCO Diagnostics, Salem, N.H.).

Immunoblot Analysis

CD8⁺ T cells (5×10⁶) were lysed with RIPA buffer containing Halt™protease and phosphatase inhibitor cocktail (Thermo Scientific,Rockford, Ill.) on ice for 30 minutes. Samples were run by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andtransferred to nitrocellulose membranes. The membranes were blockedusing buffer containing 2% BSA and 0.5% sodium azide in TBST for 1 hourand incubated with rabbit polyclonal Cyp11a1 antibody (LifespanBiosciences, Seattle, Wash.) overnight at 4° C. Horseradishperoxidase-conjugated anti-rabbit IgG (GE Healthcare, UK) was used todetect Cyp11a1 protein. Mouse monoclonal anti-β-actin antibody (Sigma,St. Louis, Mo.) was used as internal control. Immunoreactive bands ofWestern blottings were quantified by densitometric quantification ofautoradiographs using Image J (NIMH, Bethesda, Md.), and expressed asrelative Cyp11a1 normalized by β-actin.

CD8⁺ T Cell Transfection

CD8⁺ T cells were transfected with a construct encoding an shRNAspecific for mouse Cyp11a1 in the pGFP-V-RS vector (Origene, Rockville,Md.) using an Amaxa mouse T cell nucleofector kit (Amaxa/Lonza, Cologne,Germany). A sequence encoding a non-effective 29-mer scrambled shRNA inthe GFP-V-RS vector was used as control. Transfection was performed asdirected by the manufacturer (Amaxa/Lonza, Cologne, Germany) using 4 μgof plasmid and Nucleofector Program X-001. Twenty-four hours aftertransfection, cells were harvested and stimulated with SIINFEKL (1μg/ml) (SEQ ID NO:1) in medium containing 2 μM monensin for 4 hours andthen harvested for flow cytometric analysis.

Flow Cytometric Analysis

For intracellular staining, 1×10⁶/ml cells were washed twice with PBScontaining 1% BSA, stimulated with 1 μg/ml SIINFEKL in the presence of 2μM monensin at 37° C. for 4 hours. After fixation with 4%paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pa.) andpermeabilization with 0.1% saponin (Sigma, St. Louis, Mo.), cells werewashed twice with PBS containing 1% BSA, then incubated with anti-mouseCD16/CD32 (2.4 G2) (BD Bioscience, San Jose, Calif.) at 4° C. for 5minutes, then stained with FITC labeled anti-mouse IFN-γ(XMG 1.2)(eBioscience, San Diego, Calif.) or PerCP-Cy5.5 labeled anti-mouse IFN-γ(XMG 1.2) (eBioscience) and PE-labeled anti-mouse IL-13 (eBio13A)(eBioscience). For some experiments, fixed cells were stained withbiotin labeled rabbit anti-Cyp11a1/p450cc polyclonal antibody (Bioss,Woburn, Mass.) followed by PE-Cy5 labeled streptavidin (PE-Cy5-SAv)(eBioscience). Cell staining was monitored on a FACSCalibur (BDBioscience) and analyzed using Flowjo software (Tree Star, Inc, Ashland,Oreg.).

Secondary Allergen Challenge Model and Adoptive Transfer

The experimental protocol for sensitization and challenge to OVA was asdescribed previously (Oka, H. et al. Cell. Immunol. 206, 7-15 (2000);Sundrud, M. S. and Nolan, M. A. Curr. Opin. Immunol. 22, 286-292(2010)), with some modification. CD8-deficient mice were sensitized with20 μg of OVA (Calbiochem, La Jolla, Calif.) emulsified in 2.25 mg ofalum (AlumImuject; Pierce, Rockford, Ill.) on days 0 and 14 byintraperitoneal injection. Mice were challenged with 0.2% OVA for 20minutes on days 21, 22, and 23 using an ultrasonic nebulizer (modelNE-U07; Omron Healthcare, Kyoto, JP). To address the effect of Cyp11a1inhibitor on CD8⁺ T cell-mediated AHR, CD8⁻ T cells (5×10⁶) generated inmedium containing IL-2 or IL-2+AMG were injected into OVA-sensitizedCD8-deficient mice intravenously on day 37. Two hours after transfer,mice were challenged (secondary) with 1% OVA for 20 minutes. Airwayfunction was measured and samples were collected on day 38.

Assessment of Airway Function

Airway function was assessed as described previously (Oka, H. et al.Cell. Immunol. 206, 7-15 (2000); Sundrud, M. S. and Nolan, M. A. Curr.Opin. Immunol. 22, 286-292 (2010)) by measuring changes in airwayresistance (RL) in response to increasing doses of inhaled methacholine(Sigma, St. Louis, Mo.). Data were presented as percentage change fromthe baseline RL values after saline inhalation. Baseline RL values werenot significantly different among the various groups.

BAL Analysis

After measurement of AHR, lungs were lavaged via the tracheal tube with1 ml of HBSS. The supernatants were collected and IL-4, IL-5, and IL-13(eBiosicence, San Diego, Calif.) levels were measured by ELISA asdescribed previously. Total leukocyte numbers were counted anddifferentiated as described previously (Oka, H. et al. Cell. Immunol.206, 7-15 (2000); Sundrud, M. S. and Nolan, M. A. Curr. Opin. Immunol.22, 286-292 (2010)).

Immunohistochemistry Staining

CD8⁺ T cells generated in the presence of IL-2 or IL-2+IL-4 werecollected on slides. After fixing in 4% paraformaldehyde, the slideswere stained with anti-human Cyp11a1 antibody (Abcam, Cambridge, Mass.).

Mouse lungs were isolated and fixed in 10% formalin, then embedded inparaffin and cut into 5 μm thick tissue sections. Sections were stainedwith periodic acid-Schiff (PAS) and mucus-containing cells werequantitated as previously described (Oka, H. et al. Cell. Immunol. 206,7-15 (2000); Sundrud, M. S. and Nolan, M. A. Curr. Opin. Immunol. 22,286-292 (2010)). For some experiments, lung tissue expression of Cyp11a1was identified by immunohistochemistry staining using anti-human Cyp11a1antibody.

Statistical Analysis

All data were representative of at least 3 independent experiments, 4mice/group. Results were expressed as the mean±SEM. Student's two-tailedt test was used to determine the level of difference between two groups.ANOVA was used to determine the levels of difference among more than 3groups. Nonparametric analysis using the Mann-Whitney U test orKruskal-Wallis test was also used to confirm that the statisticaldifferences remained significant even if the underlying distribution wasuncertain. The p values for significance were set to 0.05 for all tests.

Example 1

This example demonstrates that Cyp11a1 mRNA, protein levels, andenzymatic activity are increased in CD8+ T cells differentiated in thepresence of IL-2+IL-4.

CD8⁺ T cells were differentiated in vitro in the presence of IL-2 orIL-2+IL-4 (FIG. 1A). Following culture for 6 days, total RNA wasextracted, cDNA was prepared and quantitative real-time PCR wasperformed. As illustrated in FIG. 1B, Cyp11a1 mRNA levels weresignificantly higher in cells differentiated in the presence of IL-4.Similarly, Cyp11a1 protein levels were elevated in these cells (FIG. 1C)as determined by densitometric quantification of immunoreactive bands onautoradiographs. Cells differentiated in IL-2 alone expressed littleCyp11a1 mRNA or protein. Immunohistochemical analysis for Cyp11a1 alsoshowed a dramatic increase in the numbers of positively stained cells incultures treated with IL-2+IL-4 compared to IL-2 alone (FIG. 1D).

The enzymatic activity of Cyp11a1 was assessed using an ELISA assay fordetection of pregnenolone levels in cell culture supernatants (Kim, C.J. et al. J. Clin. Endocrinol. Metab. 93, 696-702 (2008)). As shown inFIG. 2A, levels of pregnenolone were increased in cultures of cellsdifferentiated in IL-2 alone (1.8±0.5 pg/ml) to 424.8±35.5 pg/ml in thecells differentiated in IL-2+IL-4.

Example 2

This example demonstrates that aminoglutethimide (AMG) inhibits theenzymatic activity of Cyp11a1 without affecting mRNA or proteinexpression.

AMG is known to inhibit Cyp11a1 enzymatic activity at the initial stepof conversion of cholesterol to pregnenolone in tissues such as theadrenals (Robel, P. et al. J. Steroid Biochem. Molec. Biol. 53, 355-360(1995); Slominski, A. et al. FEBS J. 273, 2891-2901 (2006)). In cellsdifferentiated in IL-2+IL-4, addition of AMG decreased pregnenolonelevels in cell supernatants from 424.8±35.5 pg/ml to 96.4±35 pg/ml (FIG.2A). In contrast, the addition of AMG did not prevent IL-4-inducedincreases in Cyp11a1 protein levels (with or without re-stimulation withSIINFEKL (OVA₂₅₇₋₂₆₄) SEQ ID NO:1) as determined by immunoblot analysis(FIG. 2B). In fact, levels of protein were increased in AMG-treatedcells. These data suggested that the changes in pregnenolone levels wererestricted to the regulation of Cyp11a1 enzymatic activity and not dueto changes in Cyp11a1 protein levels or cell toxicity.

Example 3

This example demonstrates that Cyp11a1 enzymatic activity is essentialfor the functional conversion of CD8+ T cells from IFN-γ to IL-13producing cells.

CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 were re-stimulated withSIINFEKL (SEQ ID NO:1) and analyzed for cytokine production by flowcytometry. CD8⁺ T cells differentiated in IL-2 alone were predominantlyIFN-γ-producing with almost no IL-13-producing cells. In contrast, CD8⁺T cells differentiated in IL-2+IL-4 were predominantly IL-13 producing,with fewer cells producing IFN-γ(FIG. 2C and Table 1). To assess theimportance of the enzymatic activity of Cyp11a1 in the functionalconversion of CD8⁺ T cells, the effect of addition of the Cyp11a1 enzymeinhibitor AMG on these events was determined. CD8⁺ T cells weredifferentiated in IL-2 or IL-2+IL-4 and activated through the TCR withSIINFEKL (SEQ ID NO:1) in the presence or absence of AMG When CD8⁺ Tcells were cultured with SIINFEKL (SEQ ID NO:1) and IL-2+IL-4 in thepresence of AMG there was a dramatic dose-dependent decrease in thepercentage of IL-13-positive cells and an increase in IFN-γ-positivecells (FIG. 2C). In the presence of 500 μM AMG; the percentage ofIL-13-single-positive cells decreased from 35.7±8.2% to 14.7±8.9% andthe percentage of IFN-γ-single-positive cells increased from 14.5±5.8%to 42.4±11.5%; the percentage of IFN-γ- and IL-13-double-positive cellsincreased slightly from 8.8±2.3% to 14.7±5.5% (FIG. 2C and Table 1). Theincreased numbers of IFN-γ-positive cells in the cultures indicated thatthe drug did not have an overall suppressive or toxic effect and thatCyp11a1 enzymatic activity was indeed required for the functionalconversion of the cells to IL-13 production.

TABLE 1 IFN-γ and IL-13 expression in CD8+ T cells differentiated inIL-2 or IL-2 + IL-4 in the presence or absence of AMG

Intracellular staining of IFN-γ and IL-13 in CD8⁺ T cells with orwithout 1 μg/ml SIINFEKL or 500 μM AMG treatment. Data (mean +/− SEM)showing % positive cells were from at least 4 independent experiments.**p < 0.01 compard to the IL-2 + IL-4 + SIINFEKL group.

Example 4

This example demonstrates that silencing of Cyp11a1 with an shRNA canprevent conversion of CD8⁺ T cells from IFN-γ to IL-13-producing cells.

To further assess the requirement for Cyp11a1 activity, CD8⁺ T cellsdifferentiated in the presence of IL-2+IL-4 were transfected with agreen fluorescent protein (GFP)-encoding vector containing an shRNAconstruct specific for mouse Cyp11a1. A non-effective 29-mer scrambledshRNA in the vector was used as control. Forty-eight hours aftertransfection, the cells were stimulated with SIINFEKL(SEQ ID NO:1) for 4hours. Flow cytometric analysis for GFP indicated that there were40.2±0.7% and 43.6±1.9% GFP-positive cells following transfection ofCyp11a1-specific or scrambled shRNA, respectively. Among theGFP-positive cells, 67.8±2.8% of cells receiving the control shRNA waspositive for Cyp11a1 and this was significantly reduced to 28.8±1.2% bythe Cyp11a1-specific shRNA (FIG. 3A). After transfection of the plasmidencoding the Cyp11a1-specific shRNA, the percentage ofIFN-γ-single-positive cells increased to 26.3±1.7% compared to 13±2.7%in cells transfected with the scrambled shRNA; in parallel, thepercentage of IL-13-single-positive cells decreased from 33.7±0.6%(scrambled shRNA) to 18.7±6.3% in cells transfected with theCyp11a1-specific shRNA. The percentage of IFN-γ- andIL-13-double-positive cells increased slightly from 5.9±1.2% to 11±0.1%(FIG. 3B). These results demonstrated that reduction of Cyp11a1 inIL-2+IL-4 differentiated cells resulted in increased IFN-γ and decreasedIL-13 expression.

Example 5

This example demonstrates that lineage-specific transcription factorlevels in CD8+ T cells are unaffected by AMG treatment.

The major transcription factors regulating expression of IFN-γ and IL-13in T cells are T-bet and GATA3, respectively (Oka, H. et al. Cell.Immunol. 206, 7-15 (2000); Sundrud, M. S. and Nolan, M. A. Curr. Opin.Immunol. 22, 286-292 (2010).). Since Cyp11a1 appeared to play animportant role in controlling IFN-γ and IL-13 production in CD8⁺ Tcells, the relationship of Cyp11a1 and lineage-specific transcriptionfactor expression was examined. In cells differentiated in IL-2+IL-4,T-bet levels were decreased and GATA3 levels were increased compared tocells differentiated in IL-2 alone (FIG. 4). However, unlike cytokinelevels, there were no significant differences observed in cellsuntreated or treated with AMG These data suggested that Cyp11a1enzymatic activity exhibited regulatory activity downstream of theexpression of these lineage-specific transcription factors.

Example 6

This example demonstrates that adoptive transfer of AMG-treated CD8+cells fails to restore CD8+ T cell-mediated AHR and inflammation invivo.

The inventors have demonstrated that CD8-deficient mice develop a lowlevel of AHR and eosinophilic inflammation compared to WT mice followingsensitization and challenge, but that adoptive transfer of primed CD8⁺ Tcells differentiated in IL-2 can restore AHR, eosinophilia, and gobletcell metaplasia, suggesting in vivo conversion (Amsen, D. et al. Curr.Opin. Immunol. 21, 153-160 (2009); Miyahara, N. et al. Nature Med. 10,865-869 (2004); Ohnishi, H. et al. J. Allergy Clin. Immunol. 121,864-871 (2008)). This was confirmed following recovery of transferredCD8⁺ T cells from the lung and demonstrating their ability to produceIL-13 (National Asthma Education and Prevention Program (National HeartLung and Blood Institute) Third Expert Panel on the Management ofAsthma. National Center for Biotechnology Information (U.S.). Expertpanel report 3 guidelines for the diagnosis and management of asthma.Bethesda, Md.: National Institutes of Health National Heart Lung andBlood Institute; 2007). As shown in vitro, the in vivo conversion oftransferred CD8⁺ T cells was dependent on IL-4 (Martin, R. J. et al. J.Allergy Clin. Immunol. 119, 73-80 (2007)).

Since AMG prevented IL-4-induced functional conversion of CD8⁺ T cellsfrom IFN-γ to IL-13 producers in vitro, the inventors determined if thistreatment would attenuate restoration of lung allergic responses invivo. Initial studies determined that the effects of AMG on CD8⁺ T cellconversion, as demonstrated in FIG. 2C, could be detected for up toforty-eight hours before the cells recovered. Therefore, to ensure thatthe time frame for the in vivo experiments was consistent with theduration of AMG-mediated inhibition of Cyp11a1 enzyme activity, asecondary challenge model was used which shortens the time intervalbetween cell transfer and assay (FIG. 5A). Transfer of IL-2differentiated CD8⁺ T cells into sensitized and challenged CD8-deficientrecipients followed by secondary allergen challenge fully restored alllung allergic responses (FIGS. 5B and 5C). In contrast, transfer of CD8⁺T cells differentiated in IL-2 in the presence of AMG failed to restoreAHR or airway inflammation (FIGS. 5B and 5C). Levels of IL-4, IL-5, andIL-13 were significantly lower in the bronchoalveolar lavage (BAL) fluidof mice which received AMG-treated CD8⁻ T cells compared to untreatedcells (FIG. 5D). Lung sections were processed for histology and analyzedby PAS staining. The results showed that recipients of CD8⁺ T cellspretreated with AMG had less inflammation and significantly decreasednumbers of PAS⁺ mucus-containing goblet cells compared to those whichreceived CD8⁺ T cells that had been differentiated in the presence ofIL-2 without the enzyme inhibitor (FIG. 5E). Immunohistochemicalanalysis for Cyp11a1 protein expression in the lung sections was alsoperformed (FIG. 5F and Supplemental FIG. 1). The number ofpositively-stained cells was significantly increased after adoptivetransfer of CD8⁺ T cells differentiated in IL-2 into sensitized andchallenged recipients whereas few Cyp11a1-positive cells were detectedin recipients of AMG-treated cells, similar to numbers seen insensitized and challenged recipients that received no transferred cells.

Examples 7-11 below demonstrate Cyp11a1 enzyme activity inpeanut-induced intestinal allergy. In addition, the inventorsdemonstrate for the first time, the essential role of this enzyme in thefull development of intestinal allergic responses and that theinhibition of the enzymatic activity of Cyp11a1 attenuates CD4⁺ Th2 andTh17 differentiation and cytokine production.

Materials and Methods for Examples 7-11 Described Below

Mice

Five- to 6-week-old female Balb/c wild-type (WT) mice were purchasedfrom the Harlan Laboratory (Indianapolis, Ind.). All studies wereconducted under a protocol approved by the Institutional Animal Care andUse Committee of National Jewish Health.

Preparation of Peanut Protein

Crude peanut extract (PE) was prepared from defatted raw flours (GoldenPeanut Company, Alpharetta, Ga.) as previously described (E1). Briefly,the flour (1:10, wt/vol) was extracted in 10×PBS overnight at 4° C.After centrifugation at 30,000 g for 60 minutes, the supernatant wasfilter-sterilized, measured for protein concentration using the BCAmethod (Pierce, Rockford, Ill.), and stored as aliquots at −20° C.Endotoxin levels in PE solutions were less than 0.1 EU/ml as assessed bya Chromogenic LAL endotoxin assay kit (GeneScript, Piscataway, N.J.).

Sensitization and Intragastric Challenge

The experimental protocol for sensitization and challenge to peanut waspreviously described (Wang, M., et al. J. Allergy Clin. Immunol. 126,306-316 (2010).

Cyp11a1 Inhibitor Treatment In Vivo and In Vitro

Aminoglutethimide (AMG) was obtained from Sigma (St. Louis, Mo.). PEsensitized and challenged mice received different doses (0-20 mg/kg) ofthe inhibitor by average, based on doses previously reported (Oka, H.,et al. Cell. Immunol. 206, 7-15 (2000)) and are described as follows.

AMG was dissolved in 1 M hydrogen chloride and diluted with saline forin vivo studies or diluted with RPMI medium for in vitro study. Thefinal concentration of 1 M hydrogen chloride was less than 1% and 0.05%for in vivo and in vitro, respectively. PE sensitized and challengedmice received different doses (5, 10, 20 mg/kg) of the Cyp11a1 enzymeinhibitor (PE/PE/AMG) by means of gavage using a 22-gauge feeding needle(Fisher Scientific) twice a day during the peanut challenge phase.Controls included PE sensitized and challenged but vehicle (saline)treated (PE/PE/vehicle), or sham sensitized but PE challenged andvehicle-treated (PBS/PE/vehicle) mice.

Assessment of Peanut Intestinal Sensitivity Reactions

Clinical symptoms were evaluated as previously reported (Payne, A. H.Biol. Reprod. 42, 399-404 (1990)) and are described in the paragraphbelow.

Anaphylactic symptoms were evaluated 30 minutes after the oralchallenge, as previously reported (Li, X. M., et al. J. Allergy Clin.Immunol. 106:150-158 (2000)). Briefly, 0: no symptoms; 1: scratching andrubbing around the nose and head; 2: puffiness around the eyes andmouth, diarrhea, pilar erecti, reduced activity, and/or decreasedactivity with increased respiratory rate; 3: wheezing, laboredrespiration, and cyanosis around the mouth and the tail; 4: no activityafter prodding or tremor and convulsion; and 5: death. Scoring ofsymptoms was performed in a blinded manner by an independent observer.

Histology

The jejunum was processed and stained with periodic acid-Schiff (PAS)and chloroacetate esterase for detection of mucosal mucus-containingcells and mast cells respectively, as previously described (Wang, M., etal. J. Allergy Clin. Immunol. 126, 306-316 (2010); Tomkinson, A., et al.Am. J. Respir. Crit. Care Med. 163, 721-730 (2001)). Numbers of mucosalcells expressing Cyp11a1 were identified by immunohistochemical (IHC)staining using anti-human Cyp11a1 antibody (Abcam, Cambridge, Mass.).

Cytokines Levels in Cell Culture

Levels of IL-4, IL-13, IL-17A, and IFN-γ in cell culture supernatantswere measured by ELISA (eBioscience, San Diego, Calif.) as described bythe manufacturer.

Measurement of Peanut-Specific Antibody

Serum peanut-specific IgE, IgG1, and IgG2a levels were measured byELISA, as described previously (Payne, A. H. Biol. Reprod. 42, 399-404(1990)).

Histamine Levels in Plasma

Levels of histamine in plasma were measured using an enzyme immunoassayhistamine kit (Beckman Coulter, Fullerton, Calif.), as described by themanufacturer. The concentration of histamine was calculated from astandard curve provided by the manufacturer.

Pregnenolone Levels in Serum and Cell Culture Supernatants

Pregnenolone levels in serum and cell culture supernatants were measuredby ELISA (ALPCO Diagnostics, Salem, N.H.), as described by themanufacturer.

T-Cell Differentiation In Vitro

Differentiation of Th1, Th2, or Th17 cells was performed as previouslydescribed (40, 41) and is described in the following paragraph.

Differentiation of Th1, Th2, or Th17 cells was performed as previouslydescribed with minor changes (Ashino, S., et al. Intl. Immunol.22:503-513 (2010)). CD4⁺CD45RB⁺ T cells were isolated from naiveTCR-transgenic mice (OT II mice) spleen using a cell sorter (MoFlo XDP,Beckman Coulter). In the presence of mitomycin-C-treated spleen cells, 5μg/ml OVA₃₂₃₋₃₃₉ peptide, and the inhibitor AMG (400 μm), isolated naiveCD4 T cells were cultured with rmIL-2 (10 ng/ml, R/D Systems), rmlL-12(10 ng/ml, Peprotech), rmIFN-γ (5 ng/ml, Peprotech), and anti-IL-4 mAbs(10 μg/ml, eBioscience) to induce Th1 cell differentiation; with rmlL-2(10 ng/ml, R/D Systems), rmlL-4 (5 ng/ml, Peprotech), and anti-IFN-γ mAb(10 μg/ml, eBioscience) for differentiation of Th2 cells; and withrhIL-6 (50 ng/ml, Perotech), rhTGF-β(2 ng/ml, Peprotech), rmlL-23 (10ng/ml, Peprotech), anti-IL-4 mAb (10 μg/ml, eBioscience), and anti-IFN-γmAb (10 μg/ml, eBioscience) for differentiation of Th17 cells. After 6days of culture, the cells were washed with fresh medium andrestimulated with anti-CD3/anti-CD28 for 24 hrs for assay of cytokineproduction. The cells were collected for quantitative RT-PCR and Westernblot. In some experiments (for transduction experiment), the cells werecultured under Th1, Th2, and Th17 polarizing conditions for 5 days asdescribed in Methods.

Western Blot Analysis

Cell lysates were prepared from cultured CD4 T cells as previouslydescribed (Ashino, S., et al. Intl. Immunol. 22:503-513 (2010)) and inthe following paragraph.

Cultured cells were lysed as previously described (Ohnishi, H., et al.J. Allergy Clin. Immunol. 121:864-871 (2008)). Lysates were resolved bymeans of SDS-PAGE and transferred to nitrocellulose membranes. Proteinswere detected using antibodies specific for Cyp11a1 (LifeSpanBiosciences. Seattle, Wash.) followed by chemiluminescence detection (GEHealthcare, Little Chalfont, UK).

Quantitative Real-Time PCR

Real-time PCR was performed as previously described (Wang, M., et al. J.Allergy Clin. Immunol. 130, 932-944 (2012)) and in the followingparagraph.

RNA was extracted from jejunal tissue homogenates or from CD4 T cellscultured in vitro using Trizol (Invitrogen). cDNA was generated usingthe iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, Calif.).Quantitative real-time PCR was performed on the ABI Prism 7300 sequencedetection system (Applied Biosystems, Foster City, Calif.). All primersand probes used were purchased as Tagman Gene Expression Assays fromApplied Biosystems. Fold change was calculated using the Delta Deltacycle threshold (ΔΔC_(T)) method.

Expression Constructs

The Cyp11a1 shRNA sequence was generated using the Dharmacon siDESIGNcenter (Thermo Scientific). Cyp11a1 sense5′-TTCAATAAAGCTGATGAGTATTCAAGAGATACTCATCAGCTTTATTGATTTTTTC-3′ (SEQ IDNO:2) anti-sense5′-TCGAGAAAAAATCAATAAAGCTGATGAGTATCTCTTGAATACTCATCAGCTTTATTGA A-3′ (SEQID NO:3). Control firefly luciferase (luc) shRNA was describedpreviously (Musselman, C. A., et al. Proc. Natl. Acad. Sci. USA 109,787-792 (2012)). To construct the shRNA expression vectors,PAGE-purified and phosphorylated oligonucleotides (Integrated DNATechnologies, Coralville, Iowa) encoding Cyp11a1 shRNA were annealed andligated into a modified pQCXIP vector (Clontech, Mountain View, Calif.)expressing cyan fluorescent protein (CFP) (Musselman, C. A., et al.Proc. Natl. Acad. Sci. USA 109, 787-792 (2012)). Plasmid DNA encodingmouse Cyp11a1 and control firefly luciferase were purified usingendofree plasmid maxi kit (Qiagen, Valencia, Calif.) and sequenced (EtonBioscience, San Diego, Calif.).

Retrovirus Production and Transduction

Retrovirus production was performed as previously described (Maier, H.,et al. Nucleic Acids Res. 31, 5483-5489 (2003)). ΦNX packaging cellswere plated on poly-d-lysine-coated 100-mm dishes and cultured overnightto reach 60 to 80% confluency. Cells were co-transfected with thepCL-Eco viral packaging plasmid and plasmid DNA encoding Cyp11a1, orcontrol luc shRNA using Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions. Two dayspost-transfection, the virus-containing supernatant was collected andused to transfect cells.

Retroviral transduction of Th2 cells was performed as previouslydescribed (Pham, D., et al. J. Immunol. 189, 832-840 (2012)). SortedCD4⁺ T cells were cultured under Th2 cell differentiation conditions aspreviously reported (Wang, M., et al. J. Allergy Clin. Immunol. 130,932-944 (2012)). Cells were transduced with retroviruses (control lucshRNA or Cyp11a1 shRNA) by centrifugation in the presence of 8 μg/mlpolybrene (Sigma). Cells were expanded and analyzed on day 5.

Cell Sorting and Analysis of Gene Expression

Seventy-two hours after transduction, the cells were collected andlabeled with anti-mouse CD4 FITC (eBiosciences). CFP⁺CD4⁺ cells weresorted using a Synergy cell sorter (iCyt). Sorted cells were stimulatedwith 2 μg/ml anti-CD3/CD28 for quantitative RT-PCR and ELISA.Quantitative RT-PCR and ELISA were performed as described above.

Cell Viability and Growth

Cell viability was determined using the trypan blue dye exclusion assay.Cell growth was determined by counting the number of viable cells.

Statistical Analysis

ANOVA was used to determine the levels of difference between all groups.Comparisons for all pairs utilized the Tukey-Kramer highest significancedifference test. P values for significance were set at 0.05. All resultswere expressed as the means±SEM.

Example 7

This example demonstrates that the expression of Cyp11a1 is increased inthe small intestine of peanut sensitized and challenged mice.

The expression of Cyp11a1 mRNA and protein in the jejunum of WT Balb/cmice was examined. Following PE sensitization and challenge (FIG. 6A),Cyp11a1 mRNA expression was increased 3-fold in the jejunal homogenates(FIG. 6B). Immunohistochemical staining of jejunal tissue with anantibody specific for Cyp11a1 protein was mainly localized to the laminapropria of the small intestine (FIG. 6C). There were fewCyp11a1-positive cells in the mucosa of the small intestine of shamsensitized mice whereas numbers of Cyp11a1-positive cells weresignificantly increased in the PE sensitized and challenged mice (FIG.6D). Thus, Cyp11a1 expression was induced following sensitization andchallenge.

Example 8

This example demonstrates that the inhibition of Cyp11a1 attenuatesPE-induced allergic responses in vivo.

The effects of inhibition of Cyp11a1 enzymatic activation on theinduction of peanut allergy using an inhibitor, aminoglutethimide (AMG)was determined. AMG is known to block the enzymatic activity of Cyp11a1,thus preventing conversion of cholesterol to pregnenolone (Parajes, S.,et al. J. Clin. Endocrinol. Metab. 96, E1798-E1806 (2011)). To establishthat AMG inhibitory activity was limited to the enzymatic activity,pregnenolone levels in serum were measured following PE sensitizationand challenge. As shown in FIG. 7A, levels of pregnenolone weresignificantly increased in the serum of peanut sensitized and challengedmice (4.69±0.92 ng/ml) compared to sham sensitized but PE challengedmice (1.99±0.11 ng/ml). Levels of pregnenolone were significantlydecreased (2.98±0.60 ng/ml) in peanut sensitized and challenged micefollowing treatment with AMG (20 mg/kg). While PE sensitization andchallenge increased Cyp11a1 mRNA and numbers of Cyp11a1-positive cellsin the small intestine treatment with AMG (20 mg/kg) did not affectthese results (FIGS. 7B, 7C). These data confirm that Cyp11a1 enzymaticactivity, in parallel to mRNA and protein expression is induced bypeanut sensitization and challenge and that AMG specifically targets theenzymatic activity but not protein expression per se.

Administration of the inhibitor to sensitized mice resulted in adose-dependent inhibitory effect on intestinal allergy induction; 20mg/kg of the inhibitor fully prevented development of diarrhea andsignificantly diminished clinical symptom scores in PE sensitized andchallenged mice (FIGS. 8A, 8B). Lower doses of the inhibitor (10 mg/kg)partially inhibited diarrhea and symptom scores, whereas 5 mg/kg of theinhibitor had no observed inhibitory effects on diarrhea occurrence orclinical symptoms.

Mast cells are involved in allergic responses (Wang, M., et al. J.Allergy Clin. Immunol. 126, 306-316 (2010); Brandt, E. B., et al. J.Clin. Invest. 112, 1666-1677 (2003)) and the inventors demonstratedincreased numbers of mast cells and mucus-producing goblet cells in thesmall intestine of PE sensitized and challenged mice (FIGS. 8C, 8D andFIGS. 12 and 13). Mice treated with the Cyp11a1 inhibitor at a dose of20 mg/kg demonstrated markedly reduced numbers of mast cells as well asmucus-producing goblet cells in the mucosa of the small intestine. Todetect mast cell degranulation, plasma levels of histamine were measuredwithin 30 minutes of the last antigen challenge. Challenge of sensitizedmice resulted in detection of increased levels of histamine in plasma;following treatment with AMG (20 mg/kg), significantly lower levels ofplasma histamine were detected (FIG. 8E).

As the inhibitor was administered after sensitization, levels ofpeanut-specific IgE, IgG1, and IgG2a were unaffected by AMGadministration (FIG. 14). Together, these results demonstrate that AMGis a potent inhibitor of the enzymatic activity of Cyp11a1 in vivowithout affecting mRNA expression or protein levels of the enzyme. Thesedata demonstrate Cyp11a1's involvement in the triggering of allergicdiarrhea and symptoms, intestinal inflammation, and goblet cellmetaplasia.

Example 9

This example demonstrates that the inhibition of Cyp11a1 enzymaticactivity suppresses Th2 and Th17 cytokine production without impactingthe expression of lineage-specific transcription factors in vivo.

Th2 and Th17 cells have been implicated in the development of allergicdisorders, including asthma and food allergy (Wang, M., et al. J.Allergy Clin. Immunol. 130, 932-944 (2012); Wills-Karp, M., et al.Science 282, 2258-2261 (1998); Corren, J., et al. N. Engl. J. Med. 365,1088-1098 (2011); Kolls, J. K., & Linden, A. Immunity 21, 467-476(2004); Lajoie, S., et al. Nature Immunol. 11, 928-935 (2010)). PEsensitization and challenge increased IL4, IL13, and IL17A but not IFNGmRNA expression in the small intestine (FIG. 9A). In parallel,expression of the lineage-specific transcription factors GATA3 and RORγtmRNA were significantly increased in sensitized and challenged micewhile levels of T-bet mRNA were not altered (FIG. 9B). After treatmentwith AMG (20 mg/kg), IL4, IL13, and IL17A mRNA expression were reducedto baseline levels, but expression levels of T-bet, GATA3, or RORγt mRNAwere not affected (FIG. 9B), indicating that the effects on cytokinetranscription were mediated downstream of these transcription factors.Given that transcription factor expression was still increased inAMG-treated animals, drug toxicity as an explanation of the effects oncytokine expression was eliminated.

Example 10

This example demonstrates that the inhibition of Cyp11a1 enzymaticactivity suppresses Th2 and Th17 cell differentiation in vitro withoutaffecting lineage-specific transcription factor or Cyp11a1 expression.

Naive Th cells differentiate into Th1, Th2, and Th17 cells under thecontrol of specific polarizing cytokines and master transcriptionfactors (Zhu, J., et al. Annu. Rev. Immunol. 28, 445-489 (2010)). Theinventors demonstrate the effect of Cyp11a1 inhibition on Th celldifferentiation in vitro. Isolated CD4⁺CD45RB⁺ T cells from spleens ofnaive TCR transgenic mice (OT II mice) were cultured under Th1, Th2, andTh17 polarizing conditions in the presence or absence of the inhibitorAMG for 6 days and then stimulated with anti-CD3/anti-CD28.

Addition of AMG to cultured CD4 T cells under Th1, Th2, and Th17polarizing conditions had significant and distinct effects. In polarizedcells, Cyp11a1 mRNA expression was approximately 300-fold higher in Th2cells compared to Th1 cells and 10-fold higher in Th17 cells compared toTh1 cells (FIG. 10A). The addition of AMG (400 μM) to the cell culturesdid not suppress expression levels of Cyp11a1 mRNA or protein in thepolarized Th1, Th2, or Th17 cells (FIGS. 10B, 10C). As shown in FIG.10D, levels of pregnenolone were highest in the culture supernates frompolarized Th2 cells, with lower levels released from Th17 cells,followed by release from Th1 cells. Addition of AMG (400 μM) during thepolarization of Th cells in vitro significantly decreased levels ofpregnenolone in the culture supernates from Th2 cells but not in Th1cells. Levels in cultures of polarized Th17 cells were also reduced byAMG but the decreases did not reach statistical significance. Theseresults confirmed the findings that the inhibitory activity of AMGappeared restricted to the enzymatic activity of Cyp11a1 withoutaffecting gene transcription or translation. Further, the datademonstrated the highest levels of Cyp11a1 expression and enzymaticactivity in Th2 cells with little to no expression or activity in Th1cells.

In the culture supernatants of polarized Th2 cell cultures, levels ofIL-13 were decreased in the presence of the inhibitor (FIG. 10E); levelsof IL-17A were decreased by the inhibitor in polarized Th17 cellcultures, but levels of IFN-γ were not affected by the inhibitor inpolarized Th1 cell cultures. In parallel, the inhibitor decreased levelsof IL13 and IL17A mRNA in polarized Th2 and Th17 cells, respectively(FIG. 10F) but no significant effects of the inhibitor were detected onIFNG mRNA expression in Th1 cells. Consistent with results from the invivo studies, the inhibitor (400 μM) did not have any effect onlineage-specific transcription factor mRNA expression, T-bet, GATA3, orRORγt mRNA in polarized Th1, Th2, and Th17 cells, respectively (FIG.10F).

Example 11

This example demonstrates that shRNA-mediated silencing of Cyp11a1reduces Th2 cytokine expression.

To confirm the importance of Cyp11a1 in Th cell differentiation, theinventors used shRNA-mediated silencing of Cyp11a1 in polarized Th2cells. Polarized Th2 CD4 T cells were transduced with retrovirusesco-expressing cyan fluorescent protein (CFP) with control (luciferase)or Cyp11a1 shRNA. Seventy-two hours after transduction, CFP⁺CD4⁺ cellswere sorted and stimulated with 2 μg/ml anti-CD3/CD28 for 6 and 24hours. To confirm the effectiveness of Cyp11a1 gene silencing, theinventors demonstrated reduced levels of Cyp11a1 mRNA in Th2 CD4 T cellscompared to silencing with the control shRNA (FIG. 11A). As a result,levels of pregnenolone in supernates of cultured Th2 cells transfectedwith Cyp11a1 shRNA were significantly reduced compared to thosetransfected with control shRNA (FIG. 11B).

Levels of IL4 and IL13 mRNA were decreased in Th2 CD4 T cellstransfected with Cyp11a1 shRNA compared to those transfected withcontrol shRNA, without affecting levels of GATA3 mRNA (FIG. 11C). Inparallel, levels of IL-4 and IL-13 were reduced in supernatants of Th2CD4 T cell cultures transfected with Cyp11a1 shRNA (FIG. 11D). Theseresults demonstrate that silencing of Cyp11a1 in polarized Th2 CD4 Tcells resulted in decreased levels of IL4 and IL-13 mRNA and proteinwithout affecting GATA3 transcription. These results indicated thatCyp11a1 upregulation and activation is downstream of GATA3.

All of the documents cited herein are incorporated herein by reference.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing exemplary claims.

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What is claimed is:
 1. A method of treating an allergic disease in asubject who has, or is at risk of developing an allergic disease,comprising administering to the subject a therapeutically effectiveamount of a cytochrome P450 family 11 subfamily A polypeptide 1(Cyp11A1) inhibitor, wherein the inhibitor is a Cyp11A1 shRNA, andwherein the allergic disease is selected from the group consisting of anallergic lung disease, allergen-induced airway hyperresponsiveness,allergen-induced inflammation, asthma, and food allergy.
 2. The methodof claim 1, wherein the allergic disease is caused by one or moreproteinaceous allergens.
 3. The method of claim 1, wherein the subjecthas been sensitized to an allergen or is at risk of becoming exposed toan allergen.
 4. The method of claim 1, wherein the food allergy ispeanut allergy.
 5. The method of claim 1, wherein the allergic diseaseis an allergic lung disease.
 6. A method of inhibiting T-cellpro-allergic differentiation in a subject comprising administering tothe subject a therapeutically effective amount of a Cyp11A1 inhibitor,wherein the subject has an allergic disease, and wherein the Cyp11A1inhibitor is a Cyp11A1 shRNA.
 7. The method of claim 6, wherein theT-cell pro-allergic differentiation is CD4+ T-cells to Th2 and Th17 celldifferentiation.
 8. The method of claim 6, wherein the T-cellpro-allergic differentiation is CD8+ T-cells to Tc2 celldifferentiation.
 9. The method of claim 6, wherein the T-cellpro-allergic differentiation is IL4-induced conversion of CD8+ T-cellsinto IL-13 secreting cells.
 10. The method of claim 6, wherein theT-cell pro-allergic differentiation is IL-4 induced conversion of CD4+T-cells into IL-13 secreting cells.
 11. The method of claim 6, whereinthe allergic disease is selected from the group consisting of a allergiclung disease, allergen-induced airway hyperresponsiveness,allergen-induced inflammation, asthma, and food allergy.
 12. The methodof claim 11, wherein the allergic disease is caused by one or moreproteinaceous allergens.
 13. The method of claim 11, wherein the foodallergy is peanut allergy.
 14. The method of claim 11, wherein theallergic disease is allergic lung disease.